US10870846B2 - Cellular high throughput encapsulation for screening or selection - Google Patents

Cellular high throughput encapsulation for screening or selection Download PDF

Info

Publication number
US10870846B2
US10870846B2 US14/371,031 US201314371031A US10870846B2 US 10870846 B2 US10870846 B2 US 10870846B2 US 201314371031 A US201314371031 A US 201314371031A US 10870846 B2 US10870846 B2 US 10870846B2
Authority
US
United States
Prior art keywords
cells
solubilized
nucleic acid
detergent
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active, expires
Application number
US14/371,031
Other languages
English (en)
Other versions
US20150031549A1 (en
Inventor
Daniel Scott
Andreas Plückthun
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zurich Universitaet Institut fuer Medizinische Virologie
Original Assignee
Zurich Universitaet Institut fuer Medizinische Virologie
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zurich Universitaet Institut fuer Medizinische Virologie filed Critical Zurich Universitaet Institut fuer Medizinische Virologie
Assigned to UNIVERSITAT ZURICH reassignment UNIVERSITAT ZURICH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PLUECKTHUN, ANDREAS, SCOTT, DANIEL
Publication of US20150031549A1 publication Critical patent/US20150031549A1/en
Application granted granted Critical
Publication of US10870846B2 publication Critical patent/US10870846B2/en
Active legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis

Definitions

  • GPCR G protein-coupled receptor
  • NT neurotrophic factor
  • FACS fluorescence-activated cell sorting
  • DDM n-Dodecyl- ⁇ -D-Maltopyranoside
  • DM n-Decyl- ⁇ -D-Maltopyranoside
  • OG n-Octyl- ⁇ -D-Glucopyranoside
  • LbL Layer by Layer
  • CHAPS 3[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate/N,N-dimethyl-3-sulfo-N[3-[[3 ⁇ ,5 ⁇ ,7 ⁇ ,12 ⁇ )-3,7,12-trihydroxy-24-oxocholan-24-yl]amino]propyl]-1-propanaminium hydroxide
  • CHS cholesteryl hemisuccinate Tris salt
  • CHESS Cellular High throughput Encapsulation, Solubididimethylammonio]
  • alginate is a linear copolymer of (1-4)- ⁇ -D-mannuronate and alpha-L-guluronate.
  • Hyaluronic acid is a glycosaminoglycan.
  • polyions for practicing the invention include, without being restricted to, poly-L-lysine, carboxymethylcellulose, poly(sodium 4-styrenesulfonate), poly(allylamine hydrochloride), sodium polystyrene sulfonate, poly(styrene)-co-styrene sodium sulfonate (NaPSS), PLGA (polylactic-co-glycolic acid), polyacrylic acid or a water soluble polycationic polymer known for use in the cosmetics industry such as one of the polyquaternium list of compounds (a designation for different polycationic polymers used in the cosmetic industry; see the Wikipedia entry for “polyquaternium”).
  • the cationic treatment step precedes the anionic treatment step.
  • the initial layer depends on the properties of the template surface.
  • the surface of the cell is negatively charged due to the lipopolysaccharide (LPS) comprising the external face of the outer membrane. This makes the cell amenable to initial coating with a positively charged polymer.
  • LPS lipopolysaccharide
  • the process order can be reversed (i.e. the negatively charged layer is applied first) if a particular cell has a positively charged surface.
  • the encapsulation step proceeds over several rounds, repeating the sequence of anionic and cationic coating.
  • the cationic treatment step followed by an anionic treatment step may be repeated for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, giving rise to ever thicker capsules.
  • solubilizing the membrane of said encapsulated cells in a solubilization step, giving rise to a plurality of solubilized compartments comprises the step of exposing said plurality of encapsulated cells to a detergent in aqueous solution.
  • the method of the invention comprises
  • the solubilized compartments are contacted with a ligand to the target protein, and the ligand bears a detectable label.
  • the ligand that is contacted with the solubilized compartments is able to enter the solubilized compartment through the perforation or holes in the cell wall or outer membrane—if any is left—and through the encapsulation coated onto the cell in the encapsulation step, to probe the target protein retained inside the solubilized compartment.
  • Non-limiting examples for a ligand to practice the invention are an oligopeptide, an (allosteric) enzyme agonist or antagonist or ion channel agonist or antagonist, receptor agonist or antagonist, inverse agonist, reverse agonist and allosteric modulator.
  • the ligand may also be an enzyme substrate or a transition state analogue binding to a variant of the target protein.
  • Other non-limiting examples for ligands are specific binding molecules such as antibodies, DARPins (see US20120142611 (A1), incorporated by reference herein), FABs, nanobodies or single chain variable fragments (scFv).
  • functional proteins polypeptides
  • polypeptides may be used as ligands.
  • Cells expressing fumarate reductase or mutated fumarate reductase are encapsulated and the compartments solubilized with detergent.
  • Water soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is then added to the solubilized compartments, where it enters the compartment and any active fumarate reductase reduces the MTT to insoluble, purple coloured formazan.
  • the formazan precipitate remains inside the solubilized compartment and its presence in particular compartments can be detected and isolated using flow cytometry (FACS).
  • the detectable label is a fluorescent dye.
  • fluorescent dyes are 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), xanthene dyes such as 5- or 6-Carboxyfluorescein (5-FAM and 6-FAM) or Fluorescein, rhodamine dyes such as 5- or 6-Carboxytetramethylrhodamine (5 or 6-TAMRA), or cyanine dyes.
  • the selected sequence set is submitted to another round of selection according to the method of the invention.
  • the selected sequence set is thus subcloned and transfected into cells anew, and submitted to the sequence of encapsulation step, solubilization step, labelling step, selection step and isolation step, one or several times.
  • the repetition may be applied to the selected sequence set of the previous repetition without further manipulation of the selected sequence set.
  • the selected sequence set is mutated, for example by error prone PCR (Chen & Arnold, Proc. Nat. Acad. Sci. USA 1993, 90:5618-5622).
  • the selected sequence set is subjected to a treatment that deletes sequence tracts, recombines or shuffles sequence tracts between selected sequences, or introduces new sequence tracts randomly.
  • a treatment that deletes sequence tracts, recombines or shuffles sequence tracts between selected sequences, or introduces new sequence tracts randomly.
  • Such manipulation is equivalent to recombination in a physiological setting and enables larger “leaps” in evolutionary space.
  • both point mutation and recombination are combined.
  • the selected sequence set is diversified by amplification of said expressed nucleic acid sequences by a process introducing mutations into the amplified sequence, and/or by deletion or insertion of sequence tracts into said expressed nucleic acid sequences, and subsequently, the selected sequence set is submitted to another sequence of encapsulation step, solubilization step, labelling step, selection step and isolation step.
  • Identity in the context of the present invention is a single quantitative parameter representing the result of a sequence comparison position by position.
  • Methods of sequence comparison are known in the art; the BLAST algorithm available publicly is an example.
  • the target protein may be any protein expressed in the cell and retained in the solubilized compartment after encapsulation and solubilization.
  • One important non-limiting-example is a G-protein coupled receptor protein.
  • Other non-limiting examples include ion channels, enzymes, nuclear receptors, transcription factors and DNA/RNA-binding proteins.
  • Other examples for target proteins are specific binding molecules such as antibodies, DARPins, FABs, nanobodies or single chain variable fragments (scFv).
  • Small-molecular weight target proteins are retained in the solubilized compartments by fusion to other oligopeptides or proteins to form larger structures (e.g. a triple GFP tag), or by reduction of the effective pore size of the solubilized compartment by an increased number of polymer layers.
  • FIG. 2 shows a schematic representation of the LbL encapsulation of E. coli cells and the optimization of this method for the current invention
  • E. coli cells were encapsulated by laying down alternate layers of positively charged chitosan polymer and negatively charged alginate.
  • the amount of aggregated cells produced during the “Hillberg” LbL process was greatly reduced by the addition of EDTA (+EDTA) to the encapsulation solutions of alginate and chitosan.
  • EDTA EDTA
  • Reducing the pH of the encapsulation solutions below 7 resulted in stronger capsules that were able to resist detergent treatment.
  • FIG. 3 shows the characterization of encapsulated cells.
  • GPCR-expressing E. coli cells were encapsulated with 1 layer of chitosan and 1 layer of alginate in triplicate and analyzed with FACS.
  • the laser scattering properties of the naked cells allowed the definition of an arbitary gate enclosing 91.4% single cells.
  • 60.5% of particles detected in the encapsulated cell sample fell within this gate, with most of the remaining particles exhibiting scattering properties characteristic of larger particles.
  • FIG. 6 shows the method used to characterize selected 303 library members.
  • FIG. 7 shows the amino acid sequences of highly stable selected 303 library members.
  • the amino acid sequences of the selected receptors were aligned with parental rat NTS1, D03 and the high expressing clone C7E02. Locations of the transmembrane helices are indicated with cylinders whereas the number of mutations over D03 are shown in the ⁇ column.
  • FIG. 8 shows the selection of detergent stable ADRA1A mutants with CHESS.
  • Detergent-stable ADRA1A library members were selected with FACS using 200 nM BODIPY FL prazosin.
  • 21 selected clones were expressed individually, solubilized and assayed for ligand binding activity after 3 hours in PBS-E(DCC).
  • the top 4 receptors were solubilized in PBS-E(DCC) for 3 h at 20° C. in the absence of ligand. Solubilized receptors were captured from the supernatant with streptavidin paramagnetic beads at 4° C. for 1 h.
  • ADRA1ADCCA3 black circles
  • ADRA1ADCCG4 grey open squares
  • ADRA1ADCCD7 black crosses
  • ADRA1ADCCD8 grey open circles coated beads were either treated with 20 nM [ 3 H]prazosin for 1 h before being thermally challenged for 30 min at increasing temperatures or (c) treated with 20 nM [ 3 H]prazosin after heating in the absence of ligand.
  • No significant signal could be measured from ADRA1A- or A1A-05-coated beads when the receptors were solubilized in the absence of ligand.
  • Parallel measurements were taken for every receptor in the presence of 10 ⁇ M unlabeled prasozin as a competitor to determine the specific fluorescence signal. Data points are plotted as the mean of duplicate measurements, 100% represents the signal measured after heating at 20° C. for 30 min. Error bars indicate the standard error of the mean.
  • FIG. 9 shows the amino acid sequences of stable selected ADRA1A library members.
  • the amino acid sequences of the selected receptors were aligned with parental ADRA1A and the previously identified high expressing mutant A1A-05. Locations of the transmembrane helices are indicated with cylinders whereas the number of mutations over ADRA1A are shown in the L column.
  • the problem to be solved was that the cell would immediately disintegrate when exposed to detergent, thereby homogenizing the whole mixture of receptor mutants and plasmids, rendering the process useless for directed evolution, because the crucial genotype to phenotype linkage would be destroyed.
  • the “303 library” is a collection of rNTS1 mutants derived from D03. 30 variable positions in this library were identified from a saturation mutagenesis and high functional expression selection strategy. A further 3 amino acid substitutions were included in this library that have been reported to increase the stability of rNTS1. These 33 variable positions were recombined with wild-type residues to produce a library with a theoretical diversity of 8.6 ⁇ 10 9 individual mutants.
  • This library was previously applied to bacterial display to identify high expressing mutants (see Schlinkmann et al., J. Mol. Biol. 2012, 422(3), 414-28).
  • CHESS is a novel, rapid method for directly generating GPCRs stable to harsh detergents that are perfectly suited to biophysical analyses and crystallography screens.
  • CHESS might allow the direct selection of receptor mutants favoring the binding of G-protein mimetics such as peptides or even the G-proteins themselves, which could potentiate the direct selection of receptors stabilized in active conformations.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Virology (AREA)
  • Ecology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US14/371,031 2012-01-09 2013-01-09 Cellular high throughput encapsulation for screening or selection Active 2034-05-29 US10870846B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP12150453 2012-01-09
EP12150453.4 2012-01-09
EP12150453.4A EP2612916A1 (en) 2012-01-09 2012-01-09 Cellular high throughput encapsulation for screening or selection
PCT/EP2013/050330 WO2013104686A1 (en) 2012-01-09 2013-01-09 Cellular high throughput encapsulation for screening or selection

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/050330 A-371-Of-International WO2013104686A1 (en) 2012-01-09 2013-01-09 Cellular high throughput encapsulation for screening or selection

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/951,457 Continuation US11661676B2 (en) 2012-01-09 2020-11-18 Cellular high throughput encapsulation for screening or selection

Publications (2)

Publication Number Publication Date
US20150031549A1 US20150031549A1 (en) 2015-01-29
US10870846B2 true US10870846B2 (en) 2020-12-22

Family

ID=47563484

Family Applications (2)

Application Number Title Priority Date Filing Date
US14/371,031 Active 2034-05-29 US10870846B2 (en) 2012-01-09 2013-01-09 Cellular high throughput encapsulation for screening or selection
US16/951,457 Active US11661676B2 (en) 2012-01-09 2020-11-18 Cellular high throughput encapsulation for screening or selection

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/951,457 Active US11661676B2 (en) 2012-01-09 2020-11-18 Cellular high throughput encapsulation for screening or selection

Country Status (11)

Country Link
US (2) US10870846B2 (pl)
EP (2) EP2612916A1 (pl)
JP (1) JP6189329B2 (pl)
CN (1) CN104093838B (pl)
AU (1) AU2013208951B2 (pl)
CA (1) CA2860852C (pl)
DK (1) DK2802656T3 (pl)
ES (1) ES2611736T3 (pl)
HU (1) HUE032889T2 (pl)
PL (1) PL2802656T3 (pl)
WO (1) WO2013104686A1 (pl)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11661676B2 (en) 2012-01-09 2023-05-30 Universität Zürich Cellular high throughput encapsulation for screening or selection
US11788123B2 (en) 2017-05-26 2023-10-17 President And Fellows Of Harvard College Systems and methods for high-throughput image-based screening
US11959075B2 (en) 2014-07-30 2024-04-16 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12460250B2 (en) 2018-12-13 2025-11-04 President And Fellows Of Harvard College Amplification methods and systems for MERFISH and other applications

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3237908B1 (en) * 2014-12-22 2026-04-22 Universität Zürich Directed evolution of membrane proteins in eukaryotic cells with a cell wall
CA2997530A1 (en) * 2015-09-04 2017-03-09 The Scripps Research Institute Methods for identifying novel antibiotics and related compositions
JP2020520455A (ja) * 2017-05-11 2020-07-09 ザ フローリー インスティテュート オブ ニューロサイエンス アンド メンタル ヘルス 発現した配列の細胞スクリーニングのための真核細胞の被包
AU2020211622A1 (en) * 2019-01-25 2021-08-19 The Australian National University Encapsulated cells
WO2021099483A1 (en) 2019-11-20 2021-05-27 Leadxpro Ag Method of enabling pooled-library based nucleic acid constructs screening
WO2021216789A1 (en) * 2020-04-21 2021-10-28 University Of Maryland, College Park System, device, and method for single-cell encapsulation and culture

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003151A1 (en) 1991-08-10 1993-02-18 Medical Research Council Treatment of cell populations
US20040241759A1 (en) * 1997-06-16 2004-12-02 Eileen Tozer High throughput screening of libraries
WO2009101383A1 (en) 2008-02-11 2009-08-20 Heptares Therapeutics Limited Mutant proteins and methods for selecting them
WO2011047870A1 (en) 2009-10-22 2011-04-28 Plasticell Ltd Nested cell encapsulation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5291279B2 (ja) 2000-09-08 2013-09-18 ウニヴェルジテート・チューリッヒ 反復モジュールを含む反復タンパク質の集合体
EP2612916A1 (en) 2012-01-09 2013-07-10 Universität Zürich Cellular high throughput encapsulation for screening or selection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993003151A1 (en) 1991-08-10 1993-02-18 Medical Research Council Treatment of cell populations
US20040241759A1 (en) * 1997-06-16 2004-12-02 Eileen Tozer High throughput screening of libraries
WO2009101383A1 (en) 2008-02-11 2009-08-20 Heptares Therapeutics Limited Mutant proteins and methods for selecting them
WO2011047870A1 (en) 2009-10-22 2011-04-28 Plasticell Ltd Nested cell encapsulation

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
AMS Biotechnology "Gentle Cell Lysis with Higher Yield of Native Proteins" obtained from http://www.amsbio.com/brochures/Solulyse, created Jun. 6, 2006, modified Mar. 16, 2010. *
Biology Online Biology Dictionary; https://www.biology-online.org/dictionary/Constitutive_expression; accessed Nov. 9, 2019. *
CASIM A. SARKAR, IGOR DODEVSKI, MANCA KENIG, STEFAN DUDLI, ANJA MOHR, EMMANUEL HERMANS, ANDREAS PL�CKTHUN: "Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 39, 30 September 2008 (2008-09-30), pages 14808 - 14813, XP002676366, ISSN: 0027-8424, DOI: 10.1073/PNAS.0803103105
CASIM A. SARKAR, IGOR DODEVSKI, MANCA KENIG, STEFAN DUDLI, ANJA MOHR, EMMANUEL HERMANS, ANDREAS PL�CKTHUN: "Supporting information> Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 39, 30 September 2008 (2008-09-30), pages 1 - 22, XP002676367, ISSN: 0027-8424, DOI: 10.1073/PNAS.0803103105
Chen et al., "Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Demethylformamide", Proc. Natl. Acad. Sci. USA, vol. 90, pp. 5618-5622, Jun. 1993, Biochemistry, XP-002676370.
CHEN KEQIN, ARNOLD FRANCES H: "Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 90, no. 12, 15 June 1993 (1993-06-15), pages 5618 - 5622, XP002676370, ISSN: 0027-8424, DOI: 10.1073/pnas.90.12.5618
Dalby et al., "Strategy and Success for the Directed Evolution of Enzymes", Current Opinion in Structural Biology 2011, 21: 473-480, Science Direct, XP-002676368.
DALBY PAUL A: "Strategy and success for the directed evolution of enzymes.", STRATEGY AND SUCCESS FOR THE DIRECTED EVOLUTION OF ENZYMES., vol. 21, no. 4, 1 August 2011 (2011-08-01), pages 473 - 480, XP002676368, ISSN: 1879-033X, DOI: 10.1016/J.SBI.2011.05.003
DIASPRO A, SILVANO D, KROL S, ET AL: "Single living cell encapsulation in nano-organized polyelectrolyte shells", LANGMUIR, AMERICAN CHEMICAL SOCIETY, US, vol. 18, no. 13, 25 June 2002 (2002-06-25), US, pages 5047 - 5050, XP002621748, ISSN: 0743-7463, DOI: 10.1021/LA025646E
Diaspro et al., "Single Living Cell Encapsulation in Nano-organized Polyelectrolyte Shells", Langmuir 2002, 18, 5047-5050, Feb. 2002, XP-002621748.
Dodevski et al., "Evolution of Three Human GPCRs for Higher Expression and Stability", Journal of Molecular Biology, 2011, 408, 599-615, XP-28209045, Elsevier Ltd.
Hardeman et al. (2006) "Metagenomic approach for the isolation ofa novel low-temperature-active lipase fromuncultured bacteria of marine sediment" FEMS Microbiology Ecology 59(2):524-534. *
HILLBERG ANNA L , TABRIZIAN MARYAM: "Biorecognition through layer-by-layer polyelectrolyte assembly: In-situ hybridization on living cells", BIOMACROMOLECULES, AMERICAN CHEMICAL SOCIETY, US, vol. 7, no. 10, 1 October 2006 (2006-10-01), US, pages 2742 - 2750, XP002621749, ISSN: 1525-7797, DOI: 10.1021/BM060266J
Hillberg et al., "Biorecognition through Layer-by-Layer Polyelectrolyte Assembly: In-Situ Hybridization on Living Cells", Biomacromolecules 2006, 7, 2742-2750, XP-002621749, American Chemical Society.
IGOR DODEVSKI; ANDREAS PLÜCKTHUN;: "Evolution of Three Human GPCRs for Higher Expression and Stability", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 408, no. 4, 22 February 2011 (2011-02-22), United Kingdom, pages 599 - 615, XP028209045, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2011.02.051
Krol et al., "Encapsulated Living Cells on Microstructure Surfaces", Langmuir 2005, 21, 705-709, Sep. 2004, American Chemical Society, XP-002696260.
Miyazaki et al., "Exploring Nonnatural Evolutionary Pathways by Saturation Mutagenesis: Rapid Improvement of Protein Function", Journal of Molecular Evolution, 49: 716-720, 1999, XP-002676369.
MIYAZAKI K, ARNOLD F H: "Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function.", JOURNAL OF MOLECULAR EVOLUTION., SPRINGER VERLAG, NEW YORK, NY., US, vol. 49, no. 6, 1 December 1999 (1999-12-01), US, pages 716 - 720, XP002676369, ISSN: 0022-2844, DOI: 10.1007/PL00006593
Sarkar et al., "Directed Evolution of a G Protein-Coupled Receptor for Expression, Stability, and Binding Selectivity", PNAS, vol. 105, No. 39, Sep. 30, 2008, 14808-14813, XP-002676366, The National Academy of Sciences of the USA.
Sarkar et al., "Supporting Information" (of document listed above at No. 3), PNAS, 10.1073/pnas.0803103105, XP-002676367.
SCOTT D J, PL�CKTHUN A.: "Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells.", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 425, no. 3, 8 February 2013 (2013-02-08), United Kingdom, pages 662 - 677, XP002696261, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2012.11.015
Scott et al., "Direct Molecular Evolution of Detergent-Stable G Protein-Coupled Receptor Using Polymer Encapsulated Cells", Journal of Molecular Biology (2013) 425; pp. 662-677, Sep. 2012, XP-002696261.
SILKE KROL, MARC NOLTE, ALBERTO DIASPRO, DAVIDE MAZZA, RAFFAELLA MAGRASSI, ALESSANDRA GLIOZZI, AND ANDREAS FERY: "Encapsulated living cells on microstructured surfaces.", LANGMUIR, AMERICAN CHEMICAL SOCIETY, US, vol. 21, no. 2, 18 January 2005 (2005-01-18), US, pages 705 - 709, XP002696260, ISSN: 0743-7463, DOI: 10.1021/LA047715Q
STEMMER W. P. C.: "Rapid evolution of a protein in vitro by DNA shuffling", NATURE, MACMILLAN JOURNALS LTD, LONDON, vol. 370., 4 August 1994 (1994-08-04) - 4 August 1994 (1994-08-04), London, pages 389 - 391., XP002082182, ISSN: 0028-0836, DOI: 10.1038/370389a0
Stemmer, "Rapid Evolution of a Protein In Vitro by DNA Shuffling", Nature, vol. 370, Aug. 1994, XP-002082182.
Tate et al., "Engineering G Protein-Coupled Receptors to Facilitate their Structure Determination", Current Opinion in Structural Biology 2009, 19: 386-395, Aug. 2009, Elsevier Ltd.
Zhou et al., "Building a Thermostable Membrane Protein", The Journal of Biological Chemistry, vol. 275, No. 10, Mar. 10, 2000, pp. 6975-6979, XP-002380726.
ZHOU YUFENG ET AL: "Building a thermostable membrane protein", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 275, no. 10, 10 March 2000 (2000-03-10), pages 6975 - 6979, XP002380726, ISSN: 0021-9258, DOI: 10.1074/jbc.275.10.6975

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11661676B2 (en) 2012-01-09 2023-05-30 Universität Zürich Cellular high throughput encapsulation for screening or selection
US11959075B2 (en) 2014-07-30 2024-04-16 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12104151B2 (en) 2014-07-30 2024-10-01 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12209237B2 (en) 2014-07-30 2025-01-28 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12473546B2 (en) 2014-07-30 2025-11-18 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12522819B2 (en) 2014-07-30 2026-01-13 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US12522820B2 (en) 2014-07-30 2026-01-13 President And Fellows Of Harvard College Systems and methods for determining nucleic acids
US11788123B2 (en) 2017-05-26 2023-10-17 President And Fellows Of Harvard College Systems and methods for high-throughput image-based screening
US12460250B2 (en) 2018-12-13 2025-11-04 President And Fellows Of Harvard College Amplification methods and systems for MERFISH and other applications

Also Published As

Publication number Publication date
HK1199061A1 (zh) 2015-06-19
CN104093838B (zh) 2016-09-07
HUE032889T2 (hu) 2017-11-28
WO2013104686A1 (en) 2013-07-18
DK2802656T3 (en) 2017-01-16
JP2015503926A (ja) 2015-02-05
US20150031549A1 (en) 2015-01-29
CN104093838A (zh) 2014-10-08
CA2860852A1 (en) 2013-07-18
US20210309992A1 (en) 2021-10-07
EP2802656A1 (en) 2014-11-19
EP2612916A1 (en) 2013-07-10
EP2802656B1 (en) 2016-10-19
CA2860852C (en) 2021-01-12
AU2013208951A1 (en) 2014-08-14
AU2013208951B2 (en) 2018-02-22
PL2802656T3 (pl) 2017-05-31
ES2611736T3 (es) 2017-05-10
US11661676B2 (en) 2023-05-30
JP6189329B2 (ja) 2017-08-30

Similar Documents

Publication Publication Date Title
US11661676B2 (en) Cellular high throughput encapsulation for screening or selection
Scott et al. Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells
Srinivasan et al. Cohesion is established during DNA replication utilising chromosome associated cohesin rings as well as those loaded de novo onto nascent DNAs
AU745827B2 (en) Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes
Bernstein et al. Analyzing mRNA–protein complexes using a yeast three-hybrid system
Dodevski et al. Evolution of three human GPCRs for higher expression and stability
WO1998039483A9 (en) Methods for identifying nucleic acid sequences encoding agents that affect cellular phenotypes
CN107034228B (zh) 一种基于双分子荧光互补技术的筛选互作蛋白的方法
US20080242557A1 (en) System For Detecting Molecular Interactions
HK1199061B (en) Cellular high throughput encapsulation for screening or selection
US20020045188A1 (en) Methods for validating polypeptide targets that correlate to cellular phenotypes
JP2002516679A (ja) 有利に調節された特定成分の呈示量を含むカタログ化核酸ライブラリーの構築および使用
US20100022402A1 (en) Methods and Compositions for the In Vitro High-Throughput Detection of Protein/Protein Interactions
CN113481218B (zh) 巨型病毒或巨型噬菌体来源氨酰tRNA合成酶/tRNA正交体系构建方法及正交体系
US20220380936A1 (en) Encapsulation of eukaryotic cells for cellular screening of expressed sequences
Sheykhkarimli Sequencing protein interaction dynamics at proteome scale
MANUAL CytoTrap XR Premade Libraries
Myhrstad The Use of the Yeast Two Hybrid System to Detect CKS2 Dimerization and Interactions with CDK1 and CDK2
Jambhekar Identification and characterization of bud-localized mRNAs in Saccharomyces cerevisiae

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNIVERSITAT ZURICH, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCOTT, DANIEL;PLUECKTHUN, ANDREAS;SIGNING DATES FROM 20140714 TO 20140718;REEL/FRAME:033811/0980

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS

STPP Information on status: patent application and granting procedure in general

Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT VERIFIED

STCF Information on status: patent grant

Free format text: PATENTED CASE

FEPP Fee payment procedure

Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 4